gamma-Glutamylcysteine synthetase, the rate-limiting enzyme in the synthesis of glutathione is composed of a catalytic (Glclc) and a regulatory subunit (Glclr). The proposed work will focus on regulation of GLCLC, which is induced by a number of diverse chemicals that exhibit significantly different structural properties. One attribute common to many stress inducing agents is the generation of proteins that contain non-native thiol modifications which cause proteins to misfold, become ubiquitinated and exhibit proteasome-dependent degradation. The proposed work will test the hypothesis that GLCLC can be induced by an increased flux of misfolded proteins into the ubiquitin-proteasome pathway. Degradation of these proteins by the proteasome can result in accumulation of Nrf2; a transcription factor that regulates the GLCLC antioxidant element, ARE4 and which apparently undergoes ubiquitin independent, proteasome dependent degradation. The Specific Aims are: 1) Determine if over expression of proteins that model misfolding induce ARE-4-dependent induction of GLCLC. Cells will be stably transfected with inducible vectors that express a mutated beta-galactosidase that is thermally destabilizing, mutated bovine alpha-lactalbumin (alphaLB) that forms molten globular intermediates or native beta-galactosidase alphaLB without a leader sequence will be expressed in the cytosol. Rates of ubiquitination, proteasome-dependent degradation, expression of GLCLC, and expression of ARE4-dependent heterologous report vectors will be determined. Nrf2 dominant inhibitors will be used as well. 2) Determine if heat shock or oxidative stress utilize the same pathway for induction of GLCLC as misfolded protein. The approach used for this aim is the same as Aim 1. 3) Determine if over expression of short lived natively folded proteins induce ARE4-dependent induction of GLCLC. Cells will be stably transfected with an inducible vector that expresses either ubiquitin-proline-FLAG-beta-galactosidase or PESTodc-FLAG-beta-galactosidase. ARE4 dependent reporter vectors and Nrf2 dominant inhibitors will be used. 4) Determine if degradation of Nrf2 is ubiquitin independent but proteasome dependent. The experimental design of Aims 1 and 2 will determine if expression of GLCLC is mediated by Nrf2 binding to the ARE4 element located in the GLCLC promoter region. This aim will use a urine Elubiquitin ligase temperature sensitive mutant cell line and its control cell line to identify the degradation pathway of Nrf2. 5) Determine if Nrf2 must be phosphorylated in order to induce ARE4. This will be approached using biochemical, genetic, and pharmacological techniques.